Orally administrable compositions comprising avocado/soybean unsaponifiables and lipoic acid and methods of administration

ABSTRACT

An orally administrable composition for treating or reducing damage to connective tissue or for treating or reducing inflammatory symptoms associated with damage to connective tissue includes a synergistic combination of: (i) avocado/soybean unsaponifiables; and (ii) lipoic acid, or a salt or derivative thereof. Methods for treating or reducing damage to connective tissue, for treating or reducing inflammatory symptoms associated with damage to connective tissue, or for reducing levels of one or more inflammatory mediators in connective tissue include administering the orally administrable composition to an avian or mammalian subject.

This utility patent application is a Continuation of U.S. patentapplication Ser. No. 15/243,171 filed on Aug. 22, 2016, which claims thebenefit of U.S. patent application Ser. No. 13/112,488 filed on May 20,2011, now U.S. Pat. No. 9,421,234, the disclosures of all of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention provides methods comprising administration of: (i)avocado/soybean unsaponifiables and (ii) lipoic acid or derivativesthereof, to a mammalian or an avian subject. The present invention alsoprovides orally administrable compositions comprising avocado/soybeanunsaponifiables and lipoic acid or derivatives thereof.

BACKGROUND OF THE INVENTION

Connective tissue is the structural framework of cartilage, bone,synovium, ligament, meniscus, and tendon in articulating joints.Components of connective tissue are produced by resident cells and thensecreted to form the extracellular matrix (ECM) characteristics of thetissue. In addition to serving as structural framework, the ECM alsoplays a critical role in cell communication and function. In articularcartilage, chondrocytes are aligned in a distinct pattern within thetype II collagen ECM framework. Bone forming osteoblasts and osteocytes,as well as bone resorbing osteoclasts, are organized in mineralized typeI collagen ECM. The few fibroblast-like and macrophage-like cells in thesynovium are also held in place by ECM. Similarly, tenocytes andligament cells are assembled together within the ECM. The synthesis andbreakdown of connective tissue ECM is controlled by a network ofregulatory molecules which are also produced by the resident tissuecells. This network includes growth factors and a wide array ofmolecules known as pro-inflammatory mediators. They include cytokines,chemokines, prostaglandins and nitric oxide. These molecules exhibitmany biological activities. They can induce cell proliferation or celldeath. These substances can also induce anabolic pathways for productionof ECM or induce catabolic enzymes that can break down the ECM. Underphysiological conditions, cell survival or death, the production orbreakdown of connective tissue ECM is tightly controlled to maintainbalanced homeostasis. The production and function of regulatorymolecules is modulated by many factors including mechanical forces,physical factors such as temperature and pH, chemicals, microbes andtheir products. Under certain conditions, these factors can elicitexcessive and untimely production of regulatory molecules leading toirreparable tissue damage, loss of function and death.

Inflammation and pro-inflammatory mediators

Tissues react to mechanical, physical, chemical insults and infection byan inflammatory response. The inflammation process is known to lead torecovery, to healing, defense against infection and is usually lifepreserving. The inflammatory response in humans and animals consists oftwo phases. The initial phase is characterized by the local synthesis ofpro-inflammatory mediators such prostaglandins and leukotrienes. Theyare derived from arachidonic acid through the action of cyclooxygenasesand lipoxygenases. These pro-inflammatory mediators increase local bloodflow and enhance the permeability of endothelial cells to allowleukocyte recruitment and accumulation. Other pro-inflammatory mediatorswhich are subsequently produced include cytokines (IL-1β, TNF-α),chemokines (IL-8), and nitric oxide. In the second phase, the resolutionphase, prostaglandins generated during the initial phase activateenzymatic pathways along which arachidonic acid is converted to chemicalmediators with anti-inflammatory properties. It has been reported thatprostaglandin E₂ (PGE₂) activates the expression of 15-lipoxygenasewhich generates anti-inflammatory lipoxins from arachidonic acid. Thus,the resolution of inflammation is driven by the pro-inflammatoryresponse. These studies indicate that the initiation, progression andtermination of the inflammation process are tightly controlled.Prolonged, exaggerated inflammation has been associated with manydisorders including osteoarthritis (OA), rheumatoid arthritis (RA),Alzheimer's disease and cardiovascular disease.

In joint tissues, chondrocytes, synoviocytes, osteoblasts, osteoclasts,ligament cells, and tenocytes produce a wide array of pro-inflammatorymediators. Among these is prostaglandin E₂ (PGE₂), which is known toplay a regulatory role by inducing the production of other mediatorsincluding cytokines, nitric oxide, and connective tissue degradingmetalloproteinase (MMP) enzymes. Due to its ability to inducemetalloproteinases (MMPs), PGE₂ contributes to the breakdown ofcartilage ECM. In addition, PGE₂ promotes bone resorption and osteophyteformation. PGE₂ sensitizes nociceptors on peripheral nerve endings,thereby contributing to the development of inflammatory pain. PGE₂levels are locally regulated by the inducible cyclooxygenase-2 (COX-2)enzyme, a nitric oxide synthase in chondrocytes that inhibits cartilageand proteoglycan degradation. In pathologic conditions such asosteoarthritis, COX-2 expression is up-regulated with a concomitantincrease in PGE₂ production.

The role of other tissues in the inflammation process is also wellestablished. Inflammation of the synovial membrane is now recognized tobe a key event in cartilage degradation in osteoarthritis, particularlyduring the early stages of the disease. Synovitis is characterized byactivation of resident macrophage-like cells and fibroblast-like cellsin the synovial membrane which leads to production of excessive amountsof pro-inflammatory mediators including TNF-α, IL-1β and PGE₂. Recentevidence suggests that synovial macrophages are the main source of thecytokines in the earliest stages of osteoarthritis and that they areimportant contributors to the cartilage damage in osteoarthritisthroughout the course of the disease. Cytokines also induce productionof PGE₂ and active metalloproteinases (MMPs). It is now well acceptedthat these mediators control the balance between ECM destruction andrepair, which has made these molecules preferred targets for therapeuticintervention. Other tissues in the joint such as the subchondral bonealso produce pro-inflammatory mediators that modulate joint health.

In addition to pro-inflammatory mediators such as cytokines andprostaglandins, reactive oxygen species (ROS) have also been implicatedin joint degeneration observed in osteoarthritis. Oxidative stressinduced by ROS such as nitric oxide and hydrogen peroxide has been shownto cause chondrocyte apoptosis and cartilage ECM breakdown. Moreover,ROS have been reported to activate signal transduction pathways thatlead to an increased production of pro-inflammatory mediators includingcytokines and prostaglandins. Studies in vitro have demonstrated alinkage between the pathways involved in the production of ROS andpro-inflammatory mediators. These studies support the notion that agentscapable of inhibiting both oxidative stress and inflammation pathwayswould be particularly useful in the modulation of inflammation.

Treatment of Inflammation in Joint Tissues using Drugs

The central role of COX-2 and PGE₂ in the pathophysiology ofosteoarthritis is reflected in the widespread use of selective COX-2inhibitors and a variety of non-selective non-steroidalanti-inflammatory drugs (NSAIDs) for the treatment of the disorder.However, prolonged administration of these drugs has adverse sideeffects, including gastrointestinal pathologies and disruption ofcartilage proteoglycan metabolism. Studies in human and animal modelshave demonstrated impaired bone healing and repair with the use of COXinhibitors. Therefore, there is a need for alternative treatments forthe management of inflammation that do not center on the use of NSAIDsto inhibit the production of PGE₂ and other pro-inflammatory mediators.

Treatment of Inflammation in Joint Tissues using Nutraceuticals

Avocado/Soybean Unsaponifiables (ASU)

Many studies have documented the benefits of avocado/soybeanunsaponifiables (ASU) for promoting joint health and the management ofosteoarthritis. Clinical studies have reported beneficial effects of ASUin human and equine osteoarthritis patients as well as in experimentalanimal models of OA. The mechanisms that could account for thebeneficial effects of ASU for osteoarthritis have been studied in vitrousing bovine and human joint tissue cells. These studies showed that ASUinhibits the expression and production of cytokines, chemokines, PGE₂,nitric oxide, and MMPs. ASU also exerts anabolic effects on cartilagemetabolism by enhancing synthesis of cartilage matrix components whilesuppressing their degradation.

Earlier studies using human osteoarthritic chondrocyte cultures foundthat ASU significantly reduces the stimulating effect of IL-1β on PGE₂production. Of the two isoforms of cyclooxygenases involved inprostaglandin synthesis, COX-2 is highly inducible in response tocytokine exposure. High levels of COX-2 expression have beendemonstrated in human synovial tissue). Several studies in experimentalanimals and humans have shown that PGE₂ synthesis and COX-2 expressionare upregulated in synovial membranes in OA. Increased levels of PGE₂have been detected in synovial tissue and in synovial fibroblasts in OA.There is experimental evidence that synovial tissue is the major sourceof eicosanoids found in osteoarthritic synovial fluid. Cytokines IL-1βand TNF-α enhance synoviocyte production of PGE₂. The reported decreasein PGE₂ synthesis by ASU appears to be associated with a decrease inCOX-2 gene expression.

Lipoic Acid (LA)

Lipoic acid (LA), also known as 1,2 dithiolane-3-pentanoic acid,1,2-dithiolane-3-valeric acid, or 6,8-thioctic acid, is a potent,naturally occurring, low molecular weight antioxidant. Lipoic acid issynthesized enzymatically in the mitochondrion from octanoic acid. It isa critical cofactor of mitochondrial decarboxylation reactions and isessential for adequate ATP production. Lipoic acid exists inenantiomeric forms: R-lipoic acid (R-LA) and S-lipoic acid (S-LA). Inbiological systems, only R-LA is conjugated to lysine residues in theamide linkage. The oxidized (LA) and reduced (DHLA) forms represent apotent redox couple. The biological effect of LA include scavenging ofreactive oxygen species, regeneration of endogenous antioxidants such asglutathione and vitamin E, metal ion chelating, and repair oxidativedamage in macromolecules. Both LA and DHLA are capable of scavengingreactive oxygen species (ROS) and reactive nitrogen species (RNS), andhave the ability to prevent protein carbonyl formation. LA and DHLA canregenerate other endogenous antioxidants such as vitamin C, vitamin E,and glutathione, thereby protecting cells against oxidative stress.Recent evidence suggests that LA not only acts as a true oxidantscavenger but in addition acts as an activator of cellular stressresponse pathways.

Studies indicate that orally administered LA elicits biologicalactivities critical in the defense against oxidative stress relatedinsults. There is a growing body of evidence suggesting that orallyadministered LA is bioavailable, safe in moderate doses and elicitsseveral metabolic and clinical effects. Reported clinical benefits of LAinvolve the following disorders: diabetic polyneuropathies (Ametov etal. The sensory symptoms of diabetic polyneuropathy are improved withalpha-lipoic acid: the SYDNEY trial. Diabetes Care. 2003, 26:770-776,disorders affecting the vascular system such as hypertension,inflammation associated diseases such as coronary atherosclerosis, andcognition-neurological disorders such as Alzheimer's Disease (Hager etal., Alpha-lipoic acid as a new treatment option for Azheimer typedementia, Archives of Gerontology and Geriatrics, 2001, 32:275-282 andHager et al., Alpha-lipoic acid as a new treatment option forAlzheimer's disease—a 48 months follow-up analysis J Neural TransmSuppl. 2007, 72:189-93. However, little is known about the role of LA injoint inflammation. The effect of LA at the cellular level is diverseand its mode of action involves biologic activities such asanti-oxidation, anti-inflammation, anti-chelation and enhancement ofkinases and phosphatases.

Derivatives of lipoic acid have been described in the art. Somederivatives of lipoic acid provide improved biological activity,improved pharmacokinetic properties such as longer half-lives, improvedbioavailability, and decreased drug interaction profiles. Derivatives oflipoic acid have been described in the following publications, herebyincorporated by reference: Gruzman et al. Synthesis and characterizationof new and potent alpha-lipoic acid derivatives. Bioorganic & MedicinalChemistry, 2004, 12:1183-1190; Melagraki et al. Synthesis and evaluationof the antioxidant and anti-inflammatory activity of novelcoumarin-3-aminoamides and their alpha-lipoic acid adducts. EuropeanJournal of Medicinal Chemistry, 2009, 44:3020-3026; Gurkan et al.,Syntheses of novel indole lipoic acid derivatives and their antioxidanteffects on lipid peroxidation. Archiv der Pharmazie, 2005, 338:67-73;Ortial et al., Fluorinated amphiphilic amino acid derivatives asantioxidant carriers: a new class of protective agents. J Med Chem2006;12-2820; and Koufaki et al. Sign and synthesis of antioxidantalpha-lipoic acid hybrids. Methods Mol Biol, 2010, 594:297-309.

SUMMARY OF THE INVENTION

The present invention provides an orally administrable compositioncomprising: (i) avocado/soybean unsaponifiables (ASU) and (ii) lipoicacid or derivatives thereof. Other forms of administration, such astopical, rectal and sublingual may also be used with this composition.

The present invention also provides a method of preventing, treating,protecting, repairing or reducing damage to connective tissues orreducing symptoms associated with damage to connective tissue in anavian or mammalian subject, comprising administering to the subject: (i)avocado/soybean unsaponifiables and (ii) lipoic acid, or derivativesthereof.

The present invention additionally provides a method of reducing levelsof one or more inflammatory mediators in connective tissue, comprisingadministering to a mammalian subject: (i) avocado/soybeanunsaponifiables and (ii) lipoic acid or derivatives thereof.

Other novel features and advantages of the present invention will becomeapparent to those skilled in the art upon examination of the followingor upon learning by practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically depicts effects of avocado/soybean unsaponifiablesand lipoic acid on PCE₂ production in LPS-activated equine chondrocytecultures.

FIG. 2 graphically depicts effects of avocado/soybean unsaponifiablesand lipoic acid on PCE₂ production in H₂O₂-activated equine chondrocytecultures.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for methods comprising administration of(i) avocado/soybean unsaponifiables (ASU), and (ii) lipoic acid orderivatives thereof, to a mammalian subject. The avocado/soybeanunsaponifiables (ASU) and lipoic acid or derivatives thereof may beadministered together in one composition or dosage form, or they may beadministered separately. In preferred embodiments, the avocado/soybeanunsaponifiables (ASU) and lipoic acid or derivatives thereof, areadministered together in one composition or dosage form, or separately,within a period in which their therapeutic properties overlap,preferably within 1 hour, more preferably within 30 minutes, and mostpreferably within 5 minutes.

The term “mammalian subject” is any mammal, including, but not limitedto humans, dogs, cats, horses, cows, and camels. The term “aviansubject” refers to birds.

The term “avocado/soybean unsaponifiables (ASU)” refers to a mixture ofavocado unsaponifiables and soybean unsaponifiables. “Unsaponifiables”are compounds which do not react with alkali to form a soap. The term“avocado unsaponifiables” refers to an extract of compounds obtainedfrom any part of an avocado (genus Persea). The avocado may be anyspecies of avocado, such as but not limited to Persea americana andPersea schiedeana. The term “soybean unsaponifiables” refers to anextract of compounds obtained from any part of a soybean (Glycine max).The soybean may be any species of soybean, such as but not limited toGlycine wild.

Avocado/soybean unsaponifiables are well known in the art and aredescribed in numerous patents and publications, including but notlimited to: U.S. Pat. Nos. 6,797,289, 7,449,487, and 6,759,543; U.S.Patent Application Publication Nos. 20080176935 and 20090087503; Their,“Unsaponifiable constituents of avocado and soya oils. Treatment ofcertain forms of arthralgia,” J. Med. Lyon 53 (222): 195-8 (Feb. 1972);Trevoux, “Unsaponifiable fractions of the avocado and soybean ingynecology,”J. Gynecol. Obstet. Biol. Reprod. 6 (1): 99-105 (January1977); Lamaud et al., “Biochemical modifications of connective tissueinduced by the non-saponifiables of avocado and soy-bean oilsadministered percutaneously in the hairless rat,” Pathol. Biol. 26 (5):269-74 (May-June 1978); Boumediene et al.,“Avocado/soya unsaponifiablesenhance the expression of transforming growth factor beta 1 and beta 2in cultured articular chondrocytes,” Arthritis Rheum. 42 (1): 148-56(January 1999); Henrotin et al., “Effects of three avocado/soybeanunsaponifiable mixtures on metalloproteinases, cytokines andprostaglandin E2 production by human articular chondrocytes,” Clin.Rheumatol. 17 (1): 31-9 (1998); Maheu et al. “Symptomatic efficacy ofavocado/soybean unsaponifiables in the treatment of osteoarthritis,”Arthritis Rheum. 41 (1): 81-91 (Jan. 1998); and Blotman et al.,“Efficacyand safety of avocado/soybean unsaponifiables in the treatment ofsymptomatic osteoarthritis,” Rev. Rheum. Engl. Ed. 64 (12): 825-34 (Dec.1997), which are each incorporated by reference in their entirety. Inaddition, avocado/soybean unsaponifiables in combination with anotheringredient (glucosamine) are currently marketed in the United Statesunder the trade name AVOCA ASU®. Avocado/soybean unsaponifiables arealso marketed in Europe under the trade name PIASCLEDINE®.

Dosage calculations can be determined by those of skilled in the art byevaluating body weight, surface area and species differences. Thetypical daily dosage of avocado/soybean unsaponifiables (ASU) is about 1mg/kg to about 12 mg/kg, preferably about 2 mg/kg to about 5 mg/kg, andmore preferably about 3 mg/kg to about 4 mg/kg. In some embodiments, thetypical daily dosage is at least 5 mg for small animals, and up to 12 gfor large animals. The daily dosage refers to the total dosageadministered in a 24-hour period.

In some embodiments, the avocado/soybean unsaponifiables areadministered on a daily basis. In other embodiments, the avocado/soybeanunsaponifiables are administered less frequently, such as once everyother day or once a week or once a month. In some embodiments, theavocado/soybean unsaponifiables are administered at a daily dose ofabout 1 mg/kg to about 12 mg/kg, preferably about 2 mg/kg to about 5mg/kg, and most preferably about 3 mg/kg to about 4 mg/kg for three daysto one month, preferably for about one week, then the daily dose isdecreased to about 25% to about 90% of the initial dose, preferablyabout 50% to about 80% of the initial dose, and most preferably about60% to about 75% of the initial dose. Dosage calculations can bedetermined by those skilled in the art by evaluating body weight,surface area & species differences.

The avocado/soybean unsaponifiables may be administered at a frequencyof one time per week to five times daily, preferably once every two daysto three times daily, more preferably one to two times daily. Inpreferred embodiments, the avocado/soybean unsaponifiables areadministered once daily. The avocado/soybean unsaponifiables may betaken with or without the administration of food.

The term “lipoic acid or derivatives thereof” refers to a compound, andsalts or derivatives thereof, having the following structure:

Lipoic acid is also known as a-lipoic acid; thioctic acid;6,8-dithiooctanoic acid; and 1,2-dithiolane-3-valeric acid. Derivativesof lipoic acid include but are not limited to esters and amides oflipoic acid, conjugates of lipoic acid, and analogues of lipoic acid.Esters and amides of lipoic acid include but are not limited to5-[1,2]-dithiolan-3-yl-pentanoic acid3-(55-[1,2]-dithiolan-3-yl-pentanoyloxy)-propyl ester and5-[1,2]-dithiolan-3-yl-pentanoic acid3-(5-[1,2]-dithiolan-3-yl-pentanoylamino)-propyl-amide. Conjugates oflipoic acid include but are not limited to coumarin-lipoic acidconjugates; indole-a-lipoic acid conjugataes such as5-[1,2]dithiolan-3yl-pentanoic acid[1-(4-fluoro-benzyl)-1H-indole-5-yl]-amide; and amphiphilic lipoic acidderivatives suchN-lactobionyl-N⁸-(5-[1,2]-dithiolan-3-yl-penoyl)-L-lysinyl-1H,1H2H-perfluorooctylamide.Analogues of lipoic acid include but are not limited to1,2-diselenolane-3-pentanoic acid and 1,2-dithiolane derivatives oflipoic acid containing catechol moieties linked through heteroaromaticrings. Derivatives of lipoic acid have been described in the followingpublications, hereby incorporated by reference: Gruzman et al. Synthesisand characterization of new and potent alpha-lipoic acid derivatives.Bioorganic & Medicinal Chemistry, 2004, 12:1183-1190; Melagraki et al.Synthesis and evaluation of the antioxidant and anti-inflammatoryactivity of novel coumarin-3-aminoamides and their alpha-lipoic acidadducts. European Journal of Medicinal Chemistry, 2009, 44:3020-3026;Gurkan et al., Syntheses of novel indole lipoic acid derivatives andtheir antioxidant effects on lipid peroxidation. Archiv der Pharmazie,2005, 338:67-73; Ortial et al., Fluorinated amphiphilic amino acidderivatives as antioxidant carriers: a new class of protective agents. JMed Chem 2006;12-2820; Koufaki et al. Sign and synthesis of antioxidantalpha-lipoic acid hybrids. Methods Mol Biol, 2010, 594:297-309; Sen etal., A positively charged alpha-lipoic acid analogue with increasedcellular uptake and more potent immunomodulatory activity. BiochemBiophys Res Commun, 1998, 247:223-228; Harnett et al., Novel lipoic acidanalogues that inhibit nitric oxide synthase. Bioorg Med Chem Lett,2002, 12:1439-1442; and Acker, Syntheses of Reduced Lipoic Acid andAnalogs of Lipoic Acid, Journal of Organic Chemistry, 1963,28:2533-2536. The lipoic acid and derivatives thereof may compriseracemates, enantiomers, or mixtures thereof.

The typical daily dosage of lipoic acid or a derivative of lipoic acidcan range from about 1 mg/day to about 15,000 mg/day. The typical dailydosage of lipoic acid or derivatives of lipoic acid can range from about0.25 mg/kg/day to about 50 mg/kg/day. The daily dosage of lipoic acidrefers to the total amount of lipoic acid administered in a 24-hourperiod. The daily dosage can be provided in one or more administrations.For example, the daily dosage can be administered once daily, twicedaily, or three or more times daily. In preferred embodiments, the dailydosage is administered in one to five administrations, preferably one tothree administrations, and more preferably one or two administrations ina 24-hour period. The daily dosage may vary according to the type ofsubject. For example, in humans, the daily dosage is preferably about 25to about 2,000 mg/day, more preferably about 50 to about 1,500 mg/day,and most preferably about 100 to about 1200 mg/day, or preferably about0.5 to about 50 mg/kg/day, more preferably about 0.6 to about 40mg/kg/day, and most preferably about 1.25 to about 30 mg/kg/day. Indogs, the daily dosage is preferably about 1 to about 1,500 mg/day, morepreferably about 10 to about 750 mg/day, and most preferably about 20 toabout 600 mg/day, or preferably about 1 to about 100 mg/kg/day, morepreferably about 10 to about 60 mg/kg/day, and most preferably about 20mg/kg/day. In cats, the daily dosage is preferably about 1 to about 100mg/day, more preferably about 3 to about 50 mg/day, and most preferablyabout 3 to about 15 mg/day, or preferably about 0.5 to about 25mg/kg/day, more preferably about 1 to about 20 mg/kg/day, and mostpreferably about 3 to 15 mg/kg/day. In horses, the daily dosage ispreferably about 125 to about 15,000 mg/day, more preferably about 500to about 12,500 mg/day, and most preferably about 200 to about 10,000mg/day, or preferably about 0.5 to about 100 mg/kg/day, more preferablyabout 1 to about 50 mg/kg/day, and most preferably about 1 to 25mg/kg/day.

In some preferred embodiments, the lipoic acid or derivatives,analogues, racemates, or enantiomers of lipoic acid, or mixturesthereof, are administered for three days to one month, preferably forabout one week, then the daily dose is decreased down to 75% of theinitial dose. Dosage calculations can be determined by those skilled inthe art by evaluating body weight, surface area & species differences.

The lipoic acid or derivatives thereof may be administered at afrequency of one time per week to five times daily, preferably onceevery two days to three times daily, more preferably one to two timesdaily. In preferred embodiments, the lipoic acid or derivatives thereofare administered once daily. The lipoic acid or derivatives thereof maybe taken with or without the administration of food.

In some embodiments, the combination of (i) avocado/soybeanunsaponifiables and (ii) lipoic acid or derivatives thereof demonstratesynergy. Synergy refers to the effect wherein a combination of two ormore components provides a result which is greater than the sum of theeffects produced by the agents when used alone. In preferredembodiments, the result is statistically significant and greater thanthe additive effect. In some embodiments, the combination ofavocado/soybean unsaponifiables and lipoic acid or derivatives thereofhave a statistically significant, greater effect than each componentalone. In preferred embodiments, the combination of avocado/soybeanunsaponifiables and lipoic acid or derivatives thereof demonstratesynergy in one or more of the following: preventing, treating, repairingor reducing damage to connective tissues; reducing symptoms associatedwith damage to connective tissue in an avian or mammalian subject; andreducing levels of one or more inflammatory mediators in connectivetissue.

The present invention provides a method of preventing, treating,repairing, reducing damage, or controlling inflammation of connectivetissues, protecting cartilage, or reducing symptoms associated withdamage to connective tissue in an avian or mammalian or avian subject,comprising administering to the subject: (i) avocado/soybeanunsaponifiables and (ii) lipoic acid or derivatives thereof. The term“connective tissue” includes but not limited to cartilage, bone,synovium, ligament, meniscus, and tendon. In some embodiments, theadministration of avocado/soybean unsaponifiables and (ii) lipoic acidor derivatives thereof may prevent, treat, repair or reduce damage toconnective tissues. The damage to connective tissue may be a result ofphysical injury or may represent “wear and tear” from continual use,weight and age, for example, from osteoarthritis. Damage to connectivetissue may also result from disease such as rheumatoid arthritis,synovial disorders, infection related rheumatic diseases andinflammatory connective tissue disorders. In some embodiments, theadministration of avocado/soybean unsaponifiables and (ii) lipoic acidor derivatives thereof may reduce symptoms associated with damage toconnective tissue in an avian or mammalian subject. Symptoms associatedwith damage to connective tissue include, but are not limited to: pain,discomfort, pressure, inflammation, stiffness and/ or swelling.

The present invention also provides a method of reducing levels of oneor more inflammatory mediators in connective tissue, comprisingadministering to an avian or mammalian subject: (i) avocado/soybeanunsaponifiables and (ii) lipoic acid or derivatives thereof. Theinflammatory mediators include, but are not limited to prostaglandinssuch as prostaglandin E₂ (PGE₂), cytokines such as interleukin-1β(IL-1β) and tumor necrosis factor-α (TNF-α), chemokines, leukotrienes,nitric oxide, and reactive oxygen species.

The administration of avocado/soybean unsaponifiables and lipoic acid,or derivatives thereof may also be useful for treating, preventing, andreducing damage or reducing symptoms associated with conditionsaffecting the cardiovascular system, nervous system, musculoskeletalsystem and gastrointestinal system. The present invention also providesfor an orally administrable composition comprising: (i) avocado/soybeanunsaponifiables and (ii) lipoic acid or derivatives thereof. The orallyadministrable composition is any dosage form which can be administeredorally, such as, but not limited to: a capsule, a tablet, a powder thatcan be dispersed in a beverage, a liquid such as a solution, suspension,or emulsion, a soft gel/chew capsule, a chewable bar or other convenientdosage form such as oral liquid in a capsule, as known in the art.

The orally administrable composition may contain one or more non-activepharmaceutical ingredients (also known generally herein as“excipients”). Non-active ingredients, for example, serve to solubilize,suspend, thicken, dilute, emulsify, stabilize, preserve, protect, color,flavor, and fashion the active ingredients into an applicable andefficacious preparation that is safe, convenient, and otherwiseacceptable for use. The excipients are preferably pharmaceuticallyacceptable excipients. Examples of classes of pharmaceuticallyacceptable excipients include lubricants, buffering agents, stabilizers,blowing agents, pigments, coloring agents, flavoring agents, fillers,bulking agents, fragrances, release modifiers, adjuvants, plasticizers,flow accelerators, mold release agents, polyols, granulating agents,diluents, binders, buffers, absorbents, glidants, adhesives,anti-adherents, acidulants, softeners, resins, demulcents, solvents,surfactants, emulsifiers, elastomers and mixtures thereof.

The orally administrable compositions may further comprise one or moreactive ingredients. For example, the compositions may further compriseone or more drugs or nutritional supplements. In some embodiments, thecompositions may further comprise compounds which are beneficial toconnective tissue. Example include, but are not limited toglycosaminoglycans such as chondroitin, aminosugars such as glucosamine,methylsulfonylmethane (MSM), green tea extracts, boswellia extracts,scutellaria extracts, acacia extracts, turmeric extracts, curcumin,cetyl myristoleate complex (CMO) and egg shell membrane.

All references cited herein are incorporated by reference in theirentirety.

EXAMPLES Example 1: Effect of Avocado/Soybean Unsaponifiables (ASU) andLipoic Acid (LA) on Prostaglandin E₂ (PGE₂) Production inLipopolysaccharide (LPS) Activated Equine Chondrocyte Cultures

Chondrocytes were pre-treated with avocado/soybean unsaponifiables (ASU)at a concentration of 8.3 μg/ml and different concentrations of lipoicacid (LA) for 24 hrs, then activated with lipopolysaccharide (LPS) (1ng/ml). Lipopolysaccharide (LPS) is an endotoxin derived from thebacterial cell wall which is used as a broad inflammatory stimulus toinduce prostaglandin E₂ (PGE₂) production. After an additional 24 hrs,supernatant was collected and assayed for PGE₂ levels. Statisticalsignificance between the activated control and the pre-treated groupswere analyzed using Tukey post-hoc analysis (mean±1 SD, n=3).

The combination of avocado/soybean unsaponifiables (ASU) at aconcentration of 8.3 μg/ml and lipoic acid (LA) at concentrations of2.5, 1.25 and 0.625 μg/ml reduced PGE₂ levels significantly more thaneither ASU (<0.001) or LA (p<0.001) alone. The results are graphed inFIG. 1.

Example 2: Effect of ASU and Lipoic Acid (LA) on Prostaglandin E₂ (PGE₂)Production in Hydrogen Peroxide (H₂O₂) Activated Equine ChondrocyteCultures

Chondrocytes were pre-treated with avocado/soybean unsaponifiables (ASU)at a concentration of 8.3 μg/ml and different concentrations of lipoicacid (LA) for 24 hrs, then activated with hydrogen peroxide (500 μM).Hydrogen peroxide is a potent oxidant used to induce prostaglandin E₂(PGE₂) production. After an additional 24 hrs, supernatant was collectedand assayed for PGE₂ levels. Statistical significance between theactivated control and the pre-treated groups were analyzed using Tukeypost-hoc analysis (mean±1 SD, n=3). The combination of avocado/soybeanunsaponifiables (ASU) at a concentration of 8.3 μg/ml and lipoic acid(LA) at concentrations of 2.5 and 1.25 μg/ml reduced PGE₂ levelssignificantly more than either ASU (<0.001) or LA (p<0.05) alone. Theresults are graphed in FIG. 2.

What is claimed:
 1. An orally administrable composition for treating orreducing damage to connective tissue, or for treating or reducinginflammatory symptoms associated with damage to connective tissue,comprising a synergistic combination of: (i) avocado/soybeanunsaponifiables in an amount of from about 1 mg/kg body weight/day toabout 12 mg/kg body weight/day; and (ii) lipoic acid, or a salt orderivative thereof, in an amount of from about 0.25 mg/kg bodyweight/day to about 100 mg/kg body weight/day.
 2. The composition ofclaim 1, comprising the avocado/soybean unsaponifiables and the lipoicacid.
 3. The composition of claim 1, wherein the lipoic acid, or saltsor derivatives thereof, comprise a compound selected from the groupconsisting of: esters and amides of lipoic acid, conjugates of lipoicacid, and analogues of lipoic acid.
 4. The composition of claim 1,wherein the lipoic acid, or salts or derivatives thereof, comprise acompound selected from the group consisting of:5-[1,2]-dithiolan-3-yl-pentanoic acid3-(5-[1,2]-dithiolan-3-yl-pentanoyloxy)-propyl ester;5-[1,2]-dithiolan-3-yl-pentanoic acid3-(5-[1,2]-dithiolan-3-yl-pentanoylamino)-propyl-amide; coumarin-lipoicacid conjugates; 5-[1,2]dithiolan-3-yl-pentanoic acid[1-(4-fluoro-benzyl)-1H-indole-5-yl]-amide;N-lactobionyl-N^(ε)-(5-[1,2]-dithiolan-3 -yl-penoyl)-L-lysinyl-1H,1H,2H-perfluorooctylamide; 1,2-diselenolane-3-pentanoic acid; and1,2-dithiolane derivatives of lipoic acid containing catechol moietieslinked through heteroaromatic rings.
 5. The composition of claim 1,wherein the lipoic acid has the following structural formula:


6. A method for treating or reducing damage to connective tissue or fortreating or reducing inflammatory symptoms associated with damage toconnective tissue in an avian or mammalian subject, comprising orallyadministering to the subject an effective amount of a compositioncomprising a synergistic combination of: (i) avocado/soybeanunsaponifiables in an amount of from about 1 mg/kg body weight/day toabout 12 mg/kg body weight/day; and (ii) lipoic acid, or a salt orderivative thereof, in an amount of from about 0.25 mg/kg bodyweight/day to about 100 mg/kg body weight/day.
 7. The method of claim 6,including orally administering an effective amount of the compositioncomprising avocado/soybean unsaponifiables and lipoic acid.
 8. Themethod of claim 6, including selecting the connective tissue from thegroup consisting of: cartilage, bone, synovium, ligament, meniscus andtendon.
 9. The method of claim 6, including selecting the mammaliansubject from the group consisting of a human, a horse, a dog, a cat, acamel, or a cow.
 10. The method of claim 6, including selecting thesymptoms associated with damage to connective tissue from the groupconsisting of: pain, discomfort, pressure, inflammation, stiffnessand/or swelling.
 11. A method of reducing levels of one or moreinflammatory mediators in connective tissue, comprising orallyadministering to an avian or mammalian subject a composition in anamount effective to decrease inflammation, the composition comprising asynergistic combination of: (i) avocado/soybean unsaponifiables in anamount of from about 1 mg/kg body weight/day to about 12 mg/kg bodyweight/day; and (ii) lipoic acid, or a salt or derivative thereof, in anamount of from about 0.25 mg/kg body weight/day to about 100 mg/kg bodyweight/day.
 12. The method of claim 10, including orally administeringan effective amount of the composition comprising avocado/soybeanunsaponifiables and lipoic acid.
 13. The method of claim 10, includingselecting the one or more inflammatory mediators from the groupconsisting of: prostaglandin E₂ (PGE₂), leukotrienes, nitric oxide,cytokines, chemokines, and reactive oxygen species.
 14. The method ofclaim 10, including selecting the mammalian subject from the groupconsisting of a human, a horse, a dog, a cat, a camel or a cow.